Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although the disease is widespread with an estimated mortality rate of 59,000 people annually, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified over 100 years ago, but the slow in vitro growth rate and limited genetic tools available to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be broadly divided into two groups: free-living saprophytes and infectious pathogens. The most widely used and studied species are L. biflexa (a non-infectious saprophyte) and L. interrogans (a pathogen). However, the nonpathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we focused our attention on L. biflexa as a model to characterize conserved genes and to understand the genus as a whole, to develop new techniques, and as a heterologous host to express pathogen-specific genes in order to characterize their function. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, there are few published reports of targeted gene inactivations in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. Since L. biflexa has a better transformation frequency than other species, we endeavored to optimize new techniques in this organism. In January 2019, Dr. Philip Stewart, Lead Investigator of the Leptospirosis project, was detailed to Dr. Marshall Blooms Biology of Vector Borne-Viruses Section in the Laboratory of Virology, RML, NIAID, where he was appointed Staff Scientist in March 2019. In FY2019, no additional work was undertaken on the Leptospirosis project in anticipation of Dr. Stewarts transition to LV and departure from the Molecular Genetics Section. Reagents and strains for the Leptospira project were shared with collaborators or accompanied Dr. Stewart to LV.